LRP4 third β-propeller domain mutations cause novel congenital myasthenia by compromising agrin-mediated MuSK signaling in a position-specific manner

Abstract

Congenital myasthenic syndromes (CMS) are heterogeneous disorders in which the safety margin of neuromuscular transmission is compromised by one or more specific mechanisms. Using Sanger and exome sequencing in a CMS patient, we identified two heteroallelic mutations, p.Glu1233Lys and p.Arg1277His, in LRP4 coding for the postsynaptic low-density lipoprotein receptor-related protein 4. LRP4, expressed on the surface of the postsynaptic membrane of the neuromuscular junction, is a receptor for neurally secreted agrin, and LRP4 bound by agrin activates MuSK. Activated MuSK in concert with Dok-7 stimulates rapsyn to concentrate and anchor AChR on the postsynaptic membrane and interacts with other proteins implicated in the assembly and maintenance of the neuromuscular junction. LRP4 also functions as an inhibitor of Wnt/beta-catenin signaling. The identified mutations in LRP4 are located at the edge of its 3rd beta-propeller domain and decrease binding affinity of LRP4 for both MuSK and agrin. Mutations in the LRP4 3rd beta-propeller domain were previously reported to impair Wnt signaling and cause bone diseases including Cenani-Lenz syndactyly syndrome and sclerosteosis-2. By analyzing naturally occurring and artificially introduced mutations in the LRP4 3rd beta-propeller domain, we show that the edge of the domain regulates the MuSK signaling whereas its central cavity governs Wnt signaling. We conclude that LRP4 is a new CMS disease gene and that the 3rd beta propeller domain of LRP4 mediates the two signaling pathways in a position-specific manner.

Figure 1.

Synaptic contacts in intercostal muscle visualized by the cholinesterase reaction. (A) Normal EP. (B–D) Patient EPs. Note irregularly arrayed pleomorphic synaptic contacts at patient EPs. Bar in (A) indicates 50 μm. Bar in (D) indicates 50 μm for panels (B–D).

Figure 2.

Structure and previously identified mutations of LRP4. (A) Domain structure of LRP4 and positions of reported mutations in human, mouse and cow. p.Glu1233Lys (EK mutation) and p.Arg1277His (RH mutation) in the current studies are shown in bold. In human, LRP4 mutations cause CLSS (MIM 212780) and sclerosteosis-2 (SOST2, MIM 614305). SNPs are also associated with an increased risk for Richter syndromes (RS) and a low-trauma fracture (LTF) due to decreased bone mineral density. In mouse, mutations cause abnormal development of the apical ectodermal ridge (AER) leading to polysyndactyly and tooth abnormality, as well as abnormal developments of limbs (LIMB) and the neuromuscular junctions (NMJ). In cow, mutations lead to mulefoot disease (MFD). LRP4 harbors eight low-density lipoprotein receptor (LDLR) domain class A, four epidermal growth factor-like domains, a calcium-binding EGF-like domain, four LDLR class B repeat (β-propeller domain), a transmembrane domain and an intracellular domain. The LDLR type B repeat contains five tandem repeats of an YWTD motif to build a propeller-like structure. NPSY close to the C-terminal end is a motif for endocytosis and ESQV at the C-terminal end is a motif for binding to PDZ-containing proteins. (B) Positions of the EK and RH mutations downstream of the 4th and 5th YWTD motifs (boxed). The amino acid sequences are highly conserved across vertebrates but not in insects. Asterisks indicate strictly conserved amino acids, and dots indicate loosely conserved amino acids.

Figure 3.

p.Glu1233Lys (EK) and p.Arg1277His (RH) mutants compromise agrin-mediated upregulation of MuSK signaling but retain Wnt-suppressive activity in HEK293 cells. (A) ATF2-luciferase reporter assay of HEK293 cells to quantify agrin-mediated activation of the MuSK signaling pathway. Cells were transfected with ATF2-luc reporter and Renilla reporter plasmids along with MuSK cDNA and the indicated LRP4 cDNA. Cells were cultured with or without 10 ng/μl agrin. Wild-type (WT) LRP4-activated MuSK without agrin, and agrin further enhanced the activation. The EK and RH mutations compromise MuSK activation in the presence or absence of agrin. (B) MuSK phosphorylation assay of HEK293 cells transfected with Flag-MuSK and the indicated LRP4 cDNA with or without agrin (10 ng/μl). Phosphorylated MuSK was detected by immunoprecipitation of cell lysate by anti-phosphotyrosine antibody (p-Tyr) followed by immunoblotting with anti-FLAG antibody. Wild-type LRP4 phosphorylates MuSK, which is further enhanced by agrin, but EK and RH mutants abolish responsiveness to agrin. (C) TOPFLASH reporter assay of HEK293 cells to quantify responsiveness to Wnt3a. Cells were transfected with the TOPFLASH reporter and Renilla reporter plasmids along with the indicated LRP4 cDNA. Cells were cultured in the presence or absence of Wnt3a. Means and SD are indicated. Wild-type (WT) and mutant LRP4 (EK and RH) suppress the Wnt3a-mediated signaling to the same extent.

Figure 4.

p.Glu1233Lys (EK) and p.Arg1277His (RH) mutants compromise agrin-mediated upregulation of MuSK signaling and AChR clustering in C2C12 myoblasts/myotubes. (A) Endogenous Lrp4 expression in C2C12 myoblasts is suppressed by shRNA against mouse Lrp4 (shLrp4) by qRT-PCR. (B) MuSK phosphorylation assay of differentiation-induced C2C12 myoblasts transfected with shControl or shLrp4 and the indicated LRP4 cDNA. Phosphorylated MuSK was detected by immunoprecipitation of cell lysate by anti-phosphotyrosine antibody (p-Tyr) followed by immunoblotting with anti-MuSK antibody. Wild-type LRP4, but not EK and RH mutants, phosphorylates MuSK in Lrp4-deficient myoblasts. (C) Agrin-mediated AChR clustering in C2C12 myotubes. Myotubes are transfected with EGFP cDNA, shLRP4 and the indicated LRP4 cDNA using electroporation. AChR is visualized with Alexa594-conjugated α-bungarotoxin at 12 h after adding 10 ng/ml agrin. Right panels: Morphometric analysis showing that wild-type (WT) LRP4, but not EK and RH mutants, rescues the number and the length of AChR clusters in Lrp4-downregulated C2C12 myotubes. LRP4 has no effect on myotube length.

Figure 5.

The p.Glu1233Lys (EK) and p.Arg1277His (RH) mutants impair binding of LRP4 to MuSK and agrin. (A and B) Cell surface-binding assays. COS7 cells were transfected with the wild-type or mutant LRP4 cDNA and added with concentrated conditioned medium containing either neural Agrin-mycAP or MuSKect-mycAP as indicated. Control cells were transfected with an empty vector. Bound MuSKect-mycAP or agrin-mycAP was stained for the alkaline phosphatase activity (A). (B) The mean and SD of ALP activities of bound agrin-mycAP and MuSKect-mycAP in three independent wells. The RH and EK mutants reduce binding of MuSKect-mycAP and agrin-mycAP. (C and D) Western blotting with an anti-Flag antibody for detecting LRP4ecd-Flag; and anti-myc antibody for agrin-myc and MuSK-myc. All the transfected cDNAs were similarly expressed. (E and F) In vitro plate-binding assays. Plates were coated with the wild-type or mutant LRP4ecd-Flag protein and overlaid with purified agrin-mycAP protein (E) and MuSKect-mycAP (F). The EK and RH mutants reduce binding affinities for MuSKect-mycAP and agrin-mycAP. Mean and SE are plotted (n = 4; P< 0.05 for both MuSKect-mycAP and agrin-mycAP by two-way ANOVA).

Figure 6.

The p.Arg1170Trp (RW) and p.Trp1186Ser (WS) mutants retain the activity of agrin-mediated upregulation of MuSK signaling but compromise Wnt-suppressive activity. (A) Western blotting with an anti-Flag antibody for detecting full-length LRP4-Flag. Membrane proteins are biotinylated and precipitated with streptavidin. β-Actin proteins in each sample were detected as loading control. (B and C) ATF2-luciferase (B) and TPOFLASH (C) reporter assays of HEK293 cells to quantify activation of MuSK and Wnt signaling pathways, respectively. The RH mutant is included as a control. Means and SD of three independent experiments are indicated. (D) Cell surface-binding assays as in Figs 5A and B. Both the RW and WS mutants are able to bind to agrin-mycAP (upper) and MuSKect-mycAP (lower).

Figure 7.

The 3rd β-propeller domain of LRP4 and scheme of agrin/LRP4/MuSK complex. (A–C) Simulated three-dimensional structure of the 3rd β-propeller domain of LRP4. Positions of the analyzed mutations are indicated. The RH and EK mutations (red) are identified in our CMS patient. The RW and WS mutations (yellow) are reported in sclerosteosis-2 (12). Amino acids that are artificially mutated to alanine are shown in purple or green. Mutations at the edge of the β-propeller domain and in the central cavity are grouped together by boxes. The edge (B) and central cavity (C) of the 3rd β-propeller domain are enlarged to show locations of the naturally occurring and artificially introduced mutations. The viewing positions of (B) and (C) are not identical to (A). (D) Scheme of agrin/LRP4/MuSK. Arrows represent direct interactions: Agrin binds to the 1st EGF-like domain and the 1st β-propeller domain of LRP4 (23); MuSK binds to the 4th/5th LDLa repeats and the 3rd β-propeller domain of LRP4 (4). An artificial missense mutation in the 1st IgG-like domain of MuSK impairs binding to LRP4 (4) and deletion of this domain abolishes agrin-mediated AChR clustering (24), but the exact LRP4-binding domain(s) of MuSK remain elusive. We propose that the edge of the 3rd β-propeller domain of LRP4 is essential for binding to MuSK and for signal transduction at NMJ.

Figure 8.

Artificially engineered p.Val1252Ala (VA) and p.Ile1287Ala (IA) compromise agrin-mediated upregulation of MuSK signaling, whereas p.Asn1214Ala (NA) and p.Tyr1256Ala (YA) compromise Wnt-suppressive activity. (A) ATF2-luciferase reporter assay of HEK293 cells as in Figs 3A and 6B. The IA and VA mutations are at the edge, whereas the YA and NA mutations are in the central cavity (see Fig. 7A and Supplementary Material, Movie S1). The RH mutation in our CMS patient is included as a control. (B) TOPFLASH reporter assay of HEK293 cells as in Figs 3C and 6C. (C and D) Cell surface-binding assays as in Figs 5A, B, and 6D. The ALP activities of bound agrin-mycAP and MuSKect-mycAP in three independent wells are shown in (C). Mean and SD are indicated in (A)–(C).